BIOLOGY 
I40RARY 


NUTRITIONAL  FACTORS  IN  THE  GROWTH 
OF  CERTAIN  YEASTS  AND  BACTERIA 


By 
Louis  Freedman,....ph.  B.,  M.  S. 

NEW  YORK  CITY 
1922 


DISSERTATION 

submitted  in  partial  fulfillment  of  the  requirements  for  the  degree  of  Doctor 
of  Philosophy  in  the  Faculty  of  Pare  Science,  Columbia  University,  in  the 
City  of  New  York- 


NUTRITIONAL  FACTORS  IN  THE  GROWTH 
OF  CERTAIN  YEASTS  AND  BACTERIA 


By 
Louis  Freedman,  Ph.  B.,  M.  S. 


o 
NEW  YORK  CITY 

1922 


DISSERTATION 

submitted  in  partial  fulfillment  of  the  requirements  for  the  degree  of  Doctor 
of  Philosophy  in  the  Faculty  of  Pure  Science,  Columbia  University,  in  the 
City  of  New  York. 


;  t- 


CONTENTS. 


PART  I — VITAMINES 

Page 

Introduction    5 

Experimental  Procedure  5 

Summary  of  Conclusions 15 

Bibliography    16 

PART  II — PROTEIN  HYDROLYSATES 

Introduction    17 

Experimental  Procedure  18 

Summary  of  Conclusions   27 

Bibliography    28 


502082 


NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 


AUTHOR'S  NOTE. 

The  two  papers  that  constitute  this  dissertation  were  re- 
printed from  the  Journal  of  Metabolic  Research  (1922,  volume  I, 
No.  4,  pages  457-480),  where  they  were  published  under  the 
same  titles  in  collaboration  with  Dr.  Casimir  Funk. 


NUTRITIONAL  FACTORS  IN  THE  GROWTH  OF  CERTAIN 
YEASTS  AND  BACTERIA.f 

I.    VlTAMINES. 
By  LOUIS  FREEDMAN 

Biochemical  Laboratory  of  Columbia  University  at  the 

College  of  Physicians  and  Surgeons,  New  York  City, 
and  the  Research  Laboratory  of  H.  A.  Metz,  Ne<w  York  City. 

INTRODUCTION. 

Since  the  discovery  of  Wildier's  yeast  stimulating  "bios"  in 
1901,  rapid  progress  has  been  made  in  determining  the  nutri- 
tional requirements  of  various  yeasts  and  bacteria. 

The  early  work  of  many  investigators  has  shown  that  both 
yeasts  and  bacteria  require  for  their  growth  certain  unknown 
substances,  the  properties  of  which  correspond  closely  to  those 
of  our  present  day  water-soluble  B  vitamine.  Of  late,  however, 
considerable  discussion  has  arisen  regarding  the  identity  of  the 
substance  that  promotes  the  growth  of  yeast  cells  with  that  oi' 
vitamine  B,  and  also  its  relation  to  the  substance  that  stimulates 
the  growth  of  bacteria. 

In  the  present  work,  the  authors  have  made  an  attempt  to 
remove  some  of  the  uncertainties  regarding  these  factors,  and 
to  show  that  a  close  nutritive  analogy  exists  between  yeasts 
and  bacteria. 

EXPERIMENTAL  PROCEDURE. 

A  strain  of  hemolytic  streptococcus,  which  was  kindly  fur- 
nished by  Dr.  Mueller,**  was  used.  The  medium  for  the  growth 


t    Read    before    the    Society    for    Experimental    Biology    and    Medicine,    New    York    City, 
February   IS,    1922. 

**  The  authors  are  indebted  to  Dr.  T.  Howard  Mueller  of  the  Department  of  Bacteriology, 
College  of  Physicians  and  Surgeons,  for  the  use  of  a  strain  of  streptococcus,  and  for  a 
description  of  the  preparation  of  his  media,  and  of  the  technique  of  carrying  out  the  bac- 
teriological tests. 


6  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

of  this  organism  was  prepared  as  follows:  A  fresh  beef- 
heart,  devoid  of  fat,  and  finely  chopped,  was  boiled  for  five  min- 
utes with  an  equal  weight  of  distilled  water;  the  infusion  was 
strained  through  cloth  and  filtered,  clear,  through  filter  paper. 
This  beef-heart  infusion  was  boiled  for  15  minutes  with  two 
per  cent  of  its  weight  of  norit  charcoal;  the  decolorized  liquid 
(to  be  referred  to  hereafter  as  D.  I.)  was  filtered  clear,  and 
mixed  with  an  equal  volume  of  a  glucose-salt  solutionf  (to  be 
termed  G.  S.).  This  mixture  was  boiled  for  several  minutes, 
filtered,  adjusted  to  a  PH  of  7.4  and  sterilized  in  lots  of  250  cc. 
at  ten  pounds  pressure  for  ten  minutes.  This  medium  (referred 
to  hereafter  as  D.I.-G.S.),  inoculated  with  streptococcus,  failed 
to  give  growth  when  used  alone,  whereas  the  original  undecolor- 
ized  heart  infusion  was  favorable  for  the  growth  of  this 
organism. 

EFFECTS  OF  VARIOUS  MEDIA  ON  GROWTH  OF  STREPTOCOCCI. 

The  tests  on  streptococci  were  made  as  follows:  Duplicate 
tubes  were  prepared  containing  nine  cc.  of  the  D.I.-G.S.  solu- 
tion plus  one  cc.  of  the  solution  to  be  tested,  one  tube  being 
inoculated  and  the  other  acting  as  a  sterile  control.  One  set  of 
duplicate  tubes  of  D.I.-G.S.  alone,  and  another  set  containing  this 
solution  plus  one  per  cent  peptone,  were  used  as  additional  sterile 
and  inoculated  controls.  The  inoculations  were  made  from  a 
24-hour  bouillon  culture  previously  inoculated  from  a  pure  blood 
culture. 

The  original  undecolorized  heart  infusion  gave  a  profuse 
growth  when  inoculated  with  streptococci,  while  the  decolorized 
medium,  either  alone  or  together  with  the  glucose-salt  solution, 
failed  to  give  growth.  The  decolorized  medium,  however,  gave 
growth  when  one  percent  peptone  was  added  to  it,  while  one 
percent  peptone  solution  alone  or  together  with  glucose-salt  solu- 
tion, failed  to  give  growth.  This  confirms  some  of  the  results 
obtained  by  Mueller.1 

Autolyzed  brewers'  yeast,  diluted  20  times,  when  added  to 
the  D.I.-G.S.  medium,  supported  the  growth  of  streptococci ;  but 
in  some  cases  the  growth  was  retarded,  due  probably  to  the 
strong  acid  reaction  of  the  autolyzed  yeast.  When  the  reaction 

f    The   glucose-salt   (G.  S.)    solution  consisted   of  an   aqueous   solution  of:     NaO— 1.0%; 
C»C1«— 0.02%;    MgSO4— 0.04%;    K»HPO4— 0.20%;    Glucose   (Difco)— 0.20%. 


LOUIS  FREEDMAN  7 

was  adjusted  to  an  alkaline  range  (Pn  of  7.4-7.8),  a  more  uni- 
form growth  of  the  microorganism  was  obtained.  An  added 
factor  may  be  the  possible  presence  in  autolyzed  yeast  of  sub- 
stances which  are  toxic  to  the  specific  organism.  The  above 
results  are  given  in  detail  in  table  I. 

TABLE  I. 
Growth  of  streptococci  on  various  media. 

No.  MEDIA  Growth 

1.     10  cc.  Undecolorized   beef  -heart   infusion  ................... 


2.  10  cc.  Decolorized  beef  -heart  infusion 

3.  10  cc.  Glucose-salt   solution    (G.  S.) 

4.  10  cc.  D.  I.-G.  S.  solution 

6.  10  cc.  D.  I.-G.  S.  solution,  containing  1%  peptone 

6.  10  cc.  G.  S.  solution  containing  1%  peptone  .................         — 

7.  10  cc.  1%  peptone  solution  alone  ...........................        — 

8.  9  cc.  D.  I.-G.  S.  +  1  cc.  5%  autolyzed  yeast  ...............         + 

9.  9cc.  D.  I.-G.  S.  +  1  cc.  6%  autolyzed  yeast  (Pn  7.4-7.8)...       + 


-}-  +  +  Denotes  profuse  growth. 
4-  +  "         moderate    " 

-f  slight 

—  no 

EFFECTS  OF  THE  SAME  MEDIA  ON  THE  GROWTH  OF  YEAST  CELLS. 

For  the  test  on  the  growth  of  yeast  cells,  we  followed  the 
Funk-Dubin2  method,  which  is  the  most  convenient  and  which, 
briefly,  is  carried  out  as  follows :  A  yeast  suspension  is  prepared 
by  shaking  a  loopful  of  a  48-hour  pure  yeast  culture  in  100  cc. 
Nageli  solution  on  a  shaking  machine  for  three  hours.  Duplicate 
sets  of  tubes  are  prepared  containing 

(1)  4  cc.  yeast  suspension  +  5  cc.  Nageli  solution  +  1  cc.  water 

(2)  4  cc.  yeast  suspension  +  5  cc.  Nageli  solution  +  1  cc.  of  the 

solution  to  be  tested. 

The  tubes  are  incubated  for  20  hours  at  30° C.,  and  the 
fermentation  is  then  stopped  by  heating  the  tubes  in  a  water 
bath  to  75 °C.  The  contents  of  the  tubes  are  then  transferred  to 
special  centrifuge  tubes,  the  bottom  part  of  which  is  a  capillary 
2.5  cm.  long  and  is  graduated  in  millimeters.  These  tubes  are 
centrifuged  for  15  minutes  at  about  2500  R.P.M.,  and  the  growth 
of  the  yeast  cells  is  read  directly  on  the  tube.  The  reading  of 
tube  1,  which  constitutes  the  blank,  is  subtracted  from  that  of 
tube  2  to  give  the  net  growth  of  yeast  cells  in  millimeters.  In  all 
tests,  conditions  were  maintained  which  precluded  the  possibility 
of  bacterial  contamination. 


8  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

Undecolorized  beef-heart  infusion  gives  a  growth  of  yeast 
cells  which  compares  favorably  with  that  produced  by  a  5% 
solution  of  autolyzed  yeast  itself.  The  addition  of  glucose  to 
autolyzed  yeast  has  very  little  if  any  additional  activating  prop- 
erty, other  than  that  of  slightly  enriching  the  medium.  One  cc. 
of  decolorized  heart  infusion  when  added  to  the  Nageli  solution 
with  or  without  the  glucose-salt  solution,  has  little  or  no  effect  on 
yeast  cells;  while  the  D.I.-G.S.  medium  containing  one  percent 
of  peptone,  when  added  to  the  Nageli  solution,  stimulates  the 
growth  of  yeast  cells  just  exactly  as  much  as  when  one  percent 
peptone  solution  alone  is  added. 

TABLE  II. 

Effects  of  various  media  on  the  growth  of  yeast  cells. 

Net  yeast 

Substance  added  to  9  cc.  of  standard   Nageli  solution  Growth  in 

No.  mm. 

1.  1  cc.  5%  autolyzed  yeast  (5  CQ.  in  95  cc.  water) 10.0 

2.  Ice.  5%  autolyzed  yeast  -f-  1  cc.  10%  glucose  solution  (Difco)  11.0 

3.  1  cc.  D.  I.  solution  alone  0. 

4.  1  cc.  D.-I.-G.  S.  solution  1.5 

6.  1  cc.  1%  peptone  solution  (Difco)    2.5 

6.  1  cc.  D.  I.-G.  S.  solution  containing  1%  peptone 4.0 

7.  1  cc.  G.  S.  solution 0. 

8.  1  cc.  G.  S.  solution  containing  1%  peptone 2.0 

9.  1  cc.  Undecolorized  meat  infusion   , 7.5 

10.  1  cc.  Undecolorized  beef -heart  infusion 12.0 

It  is  apparent  from  the  results  shown  in  tables  I  and  II, 
that  there  is  present  in  beef  and  beef-heart  infusions,  and  in 
peptone  and  autolyzed  yeast,  a  substance  that  shows  compar- 
able growth-stimulation  on  hemolytic  streptococci  and  yeast  cells. 
The  question  now  naturally  arises  as  to  the  nature  of  the  sub- 
stance that  shows  such  a  marked  similarity  of  effect  on  these 
two  organisms.  From  the  standpoint  of  the  favorable  action  on 
yeast  cells,  we  are  probably  dealing  here  with  one  or  more  sub- 
stances of  the  class  of  B  vitamine.  As  for  the  growth-influencing 
action  on  streptococci,  this  either  is  due  to  a  substance  of  an 
identical  or  similar  nature;  or,  as  suggested  in  the  work  of 
Davis,3  Rivers  and  Poole,4  and  others,  we  are  dealing  with  two 
unknown  substances,  one  or  both  of  which  may  belong  to  the  class 
of  vitamines.  These  authors  found  that  certain  organisms,  par- 
ticularly those  of  the  hemophilic  type,  require  two  substances^for 


LOUIS  FREEDMAN  9 

growth,  both  of  these  substances  being  present  in  blood.  Rivers 
and  Poole4  found  that  one  of  the  substances  was  autoclave  stable, 
while  the  other  was  labile,  and  that  the  latter  could  also  be  found 
in  yeast. 

It  was  quite  logical  to  surmise,  therefore,  that  by  heating 
beef-heart  infusions  with  larger  amounts  of  norit,  we  might 
extract  a  second  substance  or  even  more  substances,  the  re- 
moval of  which  would  render  the  D.I.-G.S.  solution  inactive,  even 
on  addition  of  peptone  or  autolyzed  yeast.  Accordingly  two 
portions  of  an  undecolorized  beef-heart  infusion  were  boiled 
with  five  and  ten  percent  by  weight  of  norit  respectively  ;  but  no 
markedly  different  results  were  obtained  on  the  yeast  or  bacterial 
growth,  as  can  be  seen  by  comparing  table  III  below  with  tables 
I  and  II. 

This  second  unknown  substance  present  in  beef-heart, 
thought  to  be  in  some  way  associated  with  blood,  is  therefore 
not  related  to  the  class  of  B  vitamine,  as  it  is  not  extracted  by 
any  of  the  adsorbing  agents  which  have  an  affinity  for  this 
class  of  vitamines. 

TABLE  III. 

Growth  of  yeast  and  streptococci  on  beef-heart  infusions, 
decolorized  with  5  and  10     norit. 


Net  yeast  Bacterial 

No.  SUBSTANCE  Growth*  Growth 

in  mm. 

1.  10  cc.  Undecolorized  beef  -heart  infusion  ..........     17.5          +  +  + 

2.  6cc.  "  "  " 

4-  5  cc.  G.  S.  solution  .........................     18.0          4-  +  4- 

8.    10  cc.  Beef  -heart   infusion,    decolorized    with    5% 

norit    .......................................       1.0  — 

4.  5cc.  Beef  -heart    infusion,    decolorized    with    5% 

norit,  4-  5  cc.  G.  S.  solution  ...................       1.5  — 

5.  10  cc.  Beef  -heart  infusion,   decolorized  with   10% 

norit    .......................................       0.  — 

6.  5cc.  Beef  -heart   infusion,   decolorized  with   10% 

norit,  4-  5  cc.  G.  S.  solution  ...................       0.5  — 

7.  10  cc.  Beef  -heart    infusion,    decolorized    with    5% 

norit,  and  containing  1%  peptone  ..............       3.0  4- 

8.  5cc.  Beef  -heart    infusion,    decolorized    with    5% 

norit,  and  containing:  1%  peptone,  -f-  5  cc.  G.  S.      2.5f  4-  4- 

9.  10  cc.  Beef  -heart   infusion,   decolorized  with   10% 

norit,  and  containing  1%  peptone  .............       2.5  + 

10.       5cc.  Beef  -heart  infusion,   decolorized   with   10% 

norit,  and  containing  1%  peptone,  +  5  cc.  G.  S.      2.0f          4-  4- 


*  For  the  yeast  test,  1  cc.  of  each  of  the  above  mixtures  was  added  to  the  standard 
Nageli  solution. 

•f  The  slight  decrease  in  activity  here  is  due  to  the  smaller  amount  of  the  beef-heart 
infusion. 


10  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

SHAKING  OF  AUTOLYZED  YEAST  WITH  FULLERS  EARTH  AND  NORIT. 

Funk  and  Dubin5  have  shown  that  at  least  two  different  sub- 
stances can  be  separated  from  autolyzed  yeast  by  means  of 
shaking  with  fullers  earth.  By  this  method  it  is  now  possible  to 
separate  the  vitamine  active  for  yeast  growth,  which  has  been 
provisionally  called  "vitamine  D,"  from  the  anti-beriberi  or  B 
vitamine. 

Several  preparations  of  autolyzed  yeast  were  subjected  to 
shaking  with  various  amounts  of  both  fullers  earth  and  norit, 
as  follows :  The  clear  filtrate  of  autolyzed  yeast,  after  removal 
of  the  protein  by  heat  coagulation,  was  first  shaken  for  three 
hours  with  50  grams  of  fullers  earth  per  liter,  and  filtered. 
The  filtrate  showed  very  little  loss  of  growth-stimulating  effect 
on  yeast  cells  as  compared  with  the  original  material,  while 
there  was  no  observable  loss  of  stimulation  on  bacterial  growth. 
This  filtrate  was  again  subjected  to  shaking  with  fullers  earth, 
this  time  with  100  grams  per  liter.  The  filtrate  from  this  shak- 
ing gave  a  yeast  growth-increase  about  one  half  as  great  as  that 
given  by  the  original  5%  autolyzed  yeast  solution.  This  filtrate 
gave  as  a  rule  no  growth  of  streptococci,  although  in  one  or  two 
cases  a  slight  growth  was  obtained.  This  second  filtrate  was 
again  shaken  for  three  hours  with  100  grams  of  fullers  earth 
per  liter,  the  filtrate  from  this  final  shaking  having  almost  no 
stimulating  effect  on  yeast  growth  and  none  on  bacterial  growth. 

We  obtained  practically  similar  results  by  subjecting  auto- 
lyzed yeast  solutions  to  shaking  with  norit.  The  first  filtrate 
after  shaking  with  50  grams  of  norit  per  liter  was  still  strongly 
active  on  both  organisms,  while  the  second  filtrate,  obtained 
after  shaking  the  first  with  100  grams  per  liter,  showed  a  slight 
effect  on  yeast  growth,  but  was  entirely  negative  on  the  growth 
of  streptococci,  as  was  to  be  expected.  The  third  filtrate,  ob- 
tained after  again  shaking  the  previous  filtrate  with  100  grams 
of  norit  per  liter,  had  lost  all  activity  on  the  growth  of  both 
organisms. 

A  few  experiments  were  also  tried  with  Lloyd's  reagent,  the 
results  comparing  favorably  with  those  obtained  with  the  other 
two  adsorbing  reagents,  although  Lloyd's  reagent  is  less  ener- 
getic an  adsorber  than  fullers  earth  or  norit. 


LOUIS  FREEDMAN  11 

EXTRACTION  OF  THE  ADSORBED  MATERIAL  FROM  FULLERS  EARTH 

AND  NORIT  BY  MEANS  OF  BARYTA  AND  ACETIC  ACID 

RESPECTIVELY. 

Following  the  method  of  Seidell,6  the  fullers  earth  contain- 
ing the  adsorbed  vitamine-like  material  was  shaken  for  two 
to  three  minutes  with  one  and  a  half  volumes  of  a  10%  solution 
of  baryta  at  60  °C.  The  solid  was  then  filtered  off,  washed  with 
water,  and  the  combined  filtrate  and  washings  quickly  neutral- 
ized with  20%  sulphuric  acid.  The  filtrate,  after  removal  of  the 
barium  sulphate,  was  concentrated  in  vacuum  and  made  up  to  a 
volume  equal  to  the  original  volume  of  the  medium  before  treat- 
ment with  the  adsorbing  agent.  The  solution  was  then  adjusted 
to  a  PH  of  7.4  and  sterilized.  Unless  otherwise  noted,  one  cc.  of 
the  solution  was  used  in  all  tests. 

The  baryta  extract  of  the  fullers  earth  from  the  first  shak- 
ing with  autolyzed  yeast  (50  grams  per  liter)  showed  a  com- 
paratively strong  growth  stimulating  action  on  the  growth  of 
both  yeast  cells  and  streptococci,  as  can  be  seen  in  table  IV 
below.  This  extract  contains  nearly  all  of  the  vitamine  B  from 
autolyzed  yeast  and  a  small  part  of  the  newly  isolated  vitamine 
D.  Its  stimulating  effect  on  yeast  growth  compares  with  that 
obtained  by  Funk  and  Dubin5  as  shown  in  the  second  column 
of  table  IV.  It  also  showed  a  comparatively  strong  stimulation 
of  the  growth  of  bacteria. 

The  baryta  extract  of  the  fullers  earth  from  the  second 
shaking  with  autolyzed  yeast  (100  grams  per  liter)  had  an 
effect  on  yeast  growth  almost  equal  to  that  of  the  first  baryta 
extract;  while  the  action  on  the  growth  of  streptococci  was 
only  slightly  diminished  as  shown  by  2  (a)  and  3  (a).  This 
extract  had  no  curative  effect  on  polyneuritic  pigeons,  showing 
the  apparent  absence  of  vitamine  B.  The  fullers  earth,  during 
the  third  shaking,  failed  to  extract  any  more  of  the  active  sub- 
stance, as  its  baryta  extract  showed  no  effect  on  yeast  and 
bacterial  growth. 

Extraction  of  the  norit  ivith  acetic  acid:  Following  the 
method  of  Eddy,  Stevenson,  Johnson  and  Heft,7  the  material 
adsorbed  by  the  charcoal  was  extracted  from  it  by  heating  the 
charcoal  on  a  water  bath  for  three  hours  with  ten  parts  (by 
weight)  of  glacial  acetic  acid;  the  solution  was  filtered,  and 
evaporated  in  vacuum  to  dryness;  the  residue  was  taken  up 
in  water,  and  the  solution  again  evaporated.  This  procedure 


12 


NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 


was  repeated  several  times  to  remove  most  of  the  acid.  The 
last  traces  of  acetic  acid  were  neutralized  by  addition  of  nor- 
mal sodium  hydroxide  solution.  The  neutralized  solution  was 
made  up  to  a  volume  equal  to  the  original  volume  of  the  autolyzed 
yeast,  adjusted  to  the  proper  PH  (7.4)  and  sterilized.  One  cc. 
of  this  extract  was  used  in  each  test. 

These  acetic  acid  extracts  from  the  charcoal  gave  very  in- 
teresting results.  As  shown  in  table  IV,  Nos.  5  (a)  and  6  (a), 
they  are  practically  in  accord  with  the  results  obtained  from 
the  baryta  extracts  from  the  fullers  earth  after  adsorption  of 
the  vitamine-like  substance.  The  first  two  extracts  stimulate 
both  yeast  and  bacterial  growth,  while  the  extract  from  the 
third  shaking  [7  (a)  ]  shows  a  very  slight  stimulating  effect  on 
yeast  growth  and  no  effect  on  the  growth  of  streptococci. 

TABLE  IV. 

Effect  of  Shaking  of  Autolyzed  Yeast  with  Fullers  Earth 

and  Norit. 


No. 


FULLERS  EARTH 


1.  Autolyzed  yeast  (5%  solution) 

2.  Autolyzed    yeast    shaken    with    50 

grams  per  liter 

2  (a)  Baryta     extract    of    fullers     earth 

(from  2)  

3.  Autolyzed   yeast    (filtrate   from    2) 

shaken  with  100  grams  per  liter 

3  (a)  Baryta    extract    of    fullers     earth 

(from  3) 

4.  Autolyzed    yeast    (filtrate   from    3) 

shaken  with  100  grams  per  liter 

4  (a)  Baryta    extract    of    fullers    earth 

(from  4)  


Net  Yeast  Growth* 
in  mm.  obtained  by 
Funk  and  Our- 

Dubin  selves 

14.5  12.5 


NOHIT 


5. 


Autolyzed    yeast    shaken    with    50 
grams  per  liter 

5  (a)  Acetic    acid    extract    of    norit 

(from  5)   

6.  Autolyzed    yeast    (filtrate    from    5) 

shaken  with  100  grams  per  liter 

6  (a)  Acetic    acid    extract    of    norit 

(from  6)   

7.  Autolyzed    yeast    (filtrate    from    6) 

shaken  with  100  grams  per  liter 

7  (a)  Acetic    acid    extract    of    norit 

(from  7)   


12.0 
4.0 
6.0 
3.5 
0.5 
0. 

13.5 
3.0 
3.0 
3.0 
0.5 
0. 


9.5 

5.5 
5.0 
4.0 
0.5 
0. 

10.5 
4.0 
3.0 
3.0 
0. 
0. 


Bac- 
terial 
growth 

44- 
4-4- 
4-4 


4-4- 


4- 
4-4- 

4- 


*    Slight  variations  between  the   results   obtained  by   Funk  and   Dubin   and  by   ourselves 
are  due  to  the  use  of  different  preparations  of  autolyzed  yeast. 


LOUIS  FREEDMAN  13 

Our  results  as  detailed  in  table  IV,  show  that  we  can  obtain 
from  autolyzed  yeast  by  fractional  adsorption  with  fullers  earth 
and  norit,  and  subsequent  extraction  of  the  adsorbents,  a  con- 
centrated solution  of  vitamine  D,  almost  free  from  vitamine  B. 
This  vitamine  D  extract  is  active  in  stimulating  the  growth  of 
both  yeast  cells  and  streptococci,  but  is  not  curative  for  beriberi. 
We  were,  however,  unable  to  obtain  vitamine  B  entirely  free 
from  D  as  this  fraction  which  was  found  to  be  curative  for  beri- 
beri was  also  active  in  stimulating  the  growth  of  yeast  cells  and 
bacteria.  We  have  also  shown  that  the  extraction  of  these  active 
substances  from  the  adsorbents  with  baryta  and  acetic  acid  is 
almost  quantitative. 

SHAKING  OF  BEEF-HEART  INFUSIONS  WITH  FULLERS  EARTH  AND 

NORIT. 

A  beef-heart  infusion,  prepared  as  described  in  the  early 
part  of  this  paper,  when  shaken  for  three  hours  with  50  grams 
of  fullers  earth  per  liter,  lost  most  of  its  stimulating  effect  on 
yeast  growth,  while  it  still  retained  enough  of  the  active  sub- 
stance to  promote  the  growth  of  streptococci.  The  nitrate,  after 
a  second  shaking  with  fullers  earth,  (100  grams  per  liter) 
showed  no  activity  on  the  growth  of  either  yeast  or  bacteria. 

The  baryta  extract  of  the  fullers  earth  from  the  first  shak- 
ing, peculiarly  showed  only  a  very  slight  growth-stimulating 
activity  on  yeast;  even  the  addition  of  2  cc.  of  the  extract  to 
the  medium  increased  the  growth  of  yeast  cells  only  slightly. 
The  baryta  extract  of  the  fullers  earth  from  the  second  shaking 
was  correspondingly  less  active  on  yeast  growth,  showing  a  net 
growth  of  only  1  mm. ;  while  it  was  only  slightly  active  on  bac- 
terial growth.  The  effects  on  the  two  organisms,  however,  were 
comparable,  as  shown  in  table  V. 

Norit  removed  from  beef-heart  infusions  practically  all  of 
the  substance  which  stimulates  the  growth  of  yeast  when  the 
infusion  was  shaken  with  2%  of  its  weight  (approximately  20 
grams  per  liter)  for  three  hours.  The  infusion  was,  however, 
still  effective  for  the  growth  of  streptococci.  A  second  shaking, 
this  time  with  50  grams  per  liter,  removed  all  activity  on  yeast 
and  bacterial  growth. 

The  acetic  acid  extract  of  the  norit  from  the  first  shaking 
showed  great  growth  stimulation  on  both  organisms,  this  ex- 
tract giving  almost  as  much  stimulation  as  the  original  me- 
dium. The  net  growth  stimulating  activity  of  the  extract  on 


14  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

yeast  varied  from  5.5  to  9  mm.  with  an  average  of  about 
7.5  mm.  This  activity  compared  favorably  with  that  of  1% 
peptone  solutions.  Extraction  of  the  norit  from  the  second 
shaking  failed  to  give  any  action  on  yeast  or  bacteria.  Table  V 
gives  in  detail  the  results  of  these  extraction  experiments  with 
beef-heart  infusions. 

An  interpretation  of  these  results  shows  that  the  growth- 
stimulating  substances  are  present  in  smaller  amounts  in  beef- 
heart  infusions  than  in  autolyzed  yeast;  and  that  they  are  more 
easily  adsorbed  from  the  former  than  from  the  latter.  This 
adsorption  is  more  complete,  at  least  from  beef-heart  infusions, 
with  norit  than  with  fullers  earth.  Baryta  fails  to  extract  any 
active  substance  from  the  norit  after  adsorption,  while  glacial 
acetic  acid  is  ineffective  on  fullers  earth. 

TABLE  V. 

Effect  of  Shaking  of  Beef-Heart  Infusions  with  Fullers 
tiarth  and  Norit. 

Net  Yeast  Bac- 

No.  FULLERS  EARTH  growth  terial 

in  mm.  growth 

1.  Beef-heart  infusion  —  1  cc.   (equiv.  to  2/5 

gm.  beef -heart)   12.0  -f  -f 

2.  Beef -heart  infusion  shaken  with  50  gras.  per 

liter,  1  cc 3.0*  +  -f 

2  (a)  Beef -heart  infusion  shaken  with  50  gins,  per 

liter,  2  cc 3.5*  -}-  -f 

2  (b)  Baryta  extract  of  fullers  earth  (from  2) 

1  cc 1.0  + 

2  (c)  Baryta   extract   of   fullers    earth    (from   2) 

2  cc 1.0  -f 

3.  Beef-heart  infusion  (nitrate  from  2)  shaken 

with  100  grams  per  liter,  1  cc 0.  — 

3  (a)  Beef-heart  infusion  (nitrate  from  2)  shaken 

with  100  grams  per  liter,  2  cc 0.  — 

3  (b)  Baryta   extract    of   fullers    earth    (from    3) 

1  cc 1.0  + 

3  (c)  Baryta   extract    of   fullers    earth    (from    3) 

2  cc 1.0  + 

NORIT 

4.  Beef-heart   infusion    shaken   with   20   grams 

(2%)  per  liter,  1  cc 0.  -f 

4  (a)  Acetic    acid    extract   from    norit    (from    4) 

1  cc 7.5*  -f  + 

5.  Beef -heart  infusion  (nitrate  from  4)  shaken 

with  50  grams  per  liter,  1  cc 0.  — 

6  (a)  Acetic  acid  extract  of  norit  (from  5)  1  cc 0.  — 

•    Average  result  of  several   extraction!. 


LOUIS  FREEDMAN  15 

It  is  quite  possible  that  the  nature  and  reaction  of  the  media 
play  an  important  role  in  these  adsorption  experiments.  Inhibit- 
ing substances  may  also  be  present,  so  that  a  solution  may  be 
active  after  adsorption,  although  inactive  before  that  procedure. 
How  important  this  hypothesis  is,  we  cannot  at  present  state,  as 
we  did  not  fully  investigate  these  solutions  before  extraction  with 
the  adsorbents. 

A  summary  of  the  results  obtained  shows  that  the  substances 
which  promote  the  growth  of  bacteria  and  yeast  (as  extracted 
from  beef-heart,  peptone,  autolyzed  yeast,  etc.)  belong  to  the 
class  of  vitamines  of  the  water  soluble  B  type  ,but  are  not  identi- 
cal with  B  vitamine;  that  they  are  comparable  in  activity  and 
show  similar  properties  in  that  they  are  easily  extracted  from 
their  natural  sources  by  the  same  adsorbents,  and  are  again 
readily  recovered  from  these  adsorbents  without  appreciable  loss 
in  activity.  These  results  point  strongly  to  the  conclusion  that 
they  are  either  identical  with  vitamine  D,  or  so  similar  to  it  in 
their  physiological  behavior,  that  only  by  actual  isolation,  purifi- 
cation, and  complete  chemical  analysis  will  it  be  possible  to  differ- 
entiate them. 

The  separation  of  these  vitamine-like  substances  from  the 
bulk  of  impurities  with  which  they  are  associated  in  nature,  by 
adsorbing  them  in  fullers  earth  and  norit  and  their  subsequent 
extraction  by  chemical  treatment  of  the  adsorbents,  gives  us 
these  unknown  substances  in  a  highly  concentrated  and  com- 
paratively pure  form.  This  procedure  is  a  distinct  advantage 
over  working  with  these  substances  in  their  natural  media. 

SUMMARY  OF  CONCLUSIONS. 

1.  There  are  present  in  beef  and  beef -heart  infusions,  pep- 
tone and  autolyzed  brewer's  yeast,  certain  substances  which  show 
a  strong  growth-stimulating  activity  on  both  hemolytic  strepto- 
cocci and  yeast  cells. 

2.  These  active  substances  can  be  extracted  from  their  na- 
tural sources  by  shaking  with  certain  adsorbents,  such  as  ful- 
lers earth  and  norit  charcoal,  and  can  be  recovered  by  extract- 
ing the  adsorbents  with  baryta  and  acetic  acid  respectively. 

3.    The  properties  of  these  substances  show  them  to  be  of  a 
vitamine-like  nature ;  and  they  are  either  identical  with  the  vita- 


16  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

mine  D  described  by  Funk  and  Dubin5  or  of  a  similar  nature. 

4.  There  is  also  present  in  beef  and  beef-heart  infusions 
another  substance  which  is  necessary  for  the  growth  of  hemo- 
lytic  bacteria,  and  this  substance  is  thought  to  be  associated  with 
hemoglobin. 

BIBLIOGRAPHY  TO  PART  I. 

1.  Mueller,  J.  Howard,  Proc.  Soc.  Exp.  Biol.  Med.,  1920,  18,  14. 

2.  Funk,  C.,  and  Dubin,  H.  E.,  Jour.  Biol.  Chem.,  1920,  44,  487. 

3.  Davis,  D.  J.,  Jour.  Infec.  Dis.,  1921,  29,  171. 

4.  Rivers,  T.  M.,  and  Poole,  A.  K.,  Johns  Hopkins  Hasp.  Bull.,  1921,  32,  202. 

5.  Funk,  C.,  and  Dubin,  H.  E.,  Jour.  Biol.  Chem.,  1921,  48,  437. 

6.  Seidell,  A.,  U.  S.  Public  Health  Report,  1921,  36,  665. 

7.  Eddy,  W.  H.,  Heft,  Hattie  L.,  Stevenson,  Helen  C.,  and  Johnson,  Ruth, 

Proc.  Soc.  Exp.  Biol.  Med.,  1921,  18,  38,  Jour.  Biol.  Chem.,  1921,  47,  249. 


NUTRITIONAL  FACTORS  IN  THE  GROWTH  OF  CERTAIN 
YEASTS  AND  BACTERIA.1 

II.    PROTEIN  HYDROLYSATES. 

BY  LOUIS  FREEDMAN 

Biochemical  Laboratory  of  Columbia  University  at  the 

College  of  Physicians  and  Surgeons,  New  York  City, 
and  the  Research  Laboratory  of  H.  A.  Metz,  New  York  City. 

INTRODUCTION. 

The  use  of  proteins  and  protein  hydrolysates  for  enriching 
culture  media  for  the  .growing  of  bacteria  is  well  known;  and 
we  find  proteins  to  be  a  definite  constituent  of  many  culture 
media.  Bainbridge1  showed  that  certain  bacteria  required  pro- 
teins for  growth,  while  Robinson  and  Rettger2  used  a  casein 
hydrolysate  to  enrich  bacterial  media.  However,  the  cases  in 
which  it  has  been  definitely  shown  that  the  growth  of  the  bac- 
terial organisms  was  due  to  the  presence  of  the  protein  alone  or 
to  any  of  its  hydrolytic  cleavage  products,  are  extremely  rare. 
In  most  cases  it  has  been  shown  that  vitamines  were  present  as 
an  added  constituent  or  as  an  adhering  impurity. 

The  possible  presence  in  proteins  of  a  hitherto  unknown 
substance  which  is  now  thought  to  be  of  biological  importance 
in  nutrition,  has  of  late  been  suggested  by  several  investigators. 
In  this  connection,  Mueller3  thought  that  the  growth-stimulating 
substances  for  hemolytic  streptococci,  which  he  obtained  from 
certain  proteins,  were  definite  constituents  of  the  protein  mole- 
cule, and  he  described  the  isolation  of  two  such  substances  from 
a  preparation  of  enzyme-digested  milk  proteins  called  "ami- 
noids".  After  his  final  precipitation  of  these  substances,  how- 
ever, he  found  them,  to  be  inactive.  Goy4  also  described  the  iso- 
lation, from  certain  fungi,  of  a  nitrogen-free  acid  which  he 


1    Read    before    the    Society    of    Experimental    Biology    and    Medicine.    New    York    City, 
February  15,  1922. 


18  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

claimed  stimulated  the  growth  of  bacteria  and  yeast,  but  he  also 
found  the  finally  purified  substance  to  be  inactive. 

In  a  previous  communication5  we  have  shown  that  we  can 
obtain  from  beef  and  beef-heart  infusions,  peptone  and  autolyzed 
brewer's  yeast,  by  adsorption  with  fullers  earth  and  norit  char- 
coal, certain  substances  which  show  a  strong  growth-stimulating 
activity  on  yeast  cells  and  hemolytic  streptococci.  We  have  also 
shown  that  these  substances  can  be  extracted  from  their  com- 
bination with  fullers  earth  and  charcoal  by  treating  these  ad- 
sorbents with  baryta  and  glacial  acetic  acid  respectively.  The 
properties  of  these  substances  were  found  to  be  similar  to  those 
of  the  vitamine  D  described  by  Funk  and  Dubin6. 

In  the  following  work,  the  authors  have  tried  to  show  the 
relationship  between  the  substances  found  in  proteins,  and  vita- 
mine  D;  and  that  these  substances  are  not  present  in  purified 
proteins  in  general  but  are  found  associated  only  with  certain 
proteins. 

EXPERIMENTAL  PROCEDURE. 

For  this  work  we  have  prepared  a  series  of  well  known  pro- 
teins, consisting  of  twelve  animal  and  ten  vegetable  proteins. 
The  proteins  of  animal  origin  varied  to  a  great  extent,  and  in- 
cluded the  milk  proteins,  casein  and  lactalbumin,  three  ox-blood 
proteins,  three  egg  proteins,  muscle  protein,  liver  albumin  and 
globulin,  and  gelatin. 

For  the  proteins  of  vegetable  origin,  we  have  prepared 
edestin  (from  hemp  seed) ,  the  wheat  and  corn  proteins,  the  three 
proteins  from  the  pea,  oryzenin  (from  rice),  hordein  (from 
barley) ,  and  yeast  protein.  These  proteins  were  prepared  accord- 
ing to  the  standard  published  methods,  particular  care  being 
taken  to  have  the  proteins,  in  so  far  as  it  was  possible,  free  from 
vitamines. 

HYDROLYSIS  OF  THE  PROTEINS. 

All  of  the  above  proteins  were  hydrolysed  by  boiling  them 
with  seven  parts  of  concentrated  hydrochloric  acid  (specific 
gravity  1.19)  for  eight  to  ten  hours.  After  cooling,  the  humin 


LOUIS  FREEDMAN  19 

substance  was  filtered  off  and  the  filtrate  evaporated  to  dry- 
ness  in  vacuum.  The  residue  was  taken  up  in  water  and  the 
solution  again  evaporated  to  dryness.  This  operation  was  re- 
peated twice,  making  four  evaporations,  to  remove  the  bulk  of 
hydrochloric  acid.  The  remaining  acid  was  then  neutralized  by 
addition  of  normal  sodium  hydroxide ;  the  solution  was  made  up 
to  a  volume  corresponding  to  a  10%  solution  of  the  protein,  ad- 
justed to  a  PH  of  about  7.3  and  sterilized  at  ten  pounds  pressure 
for  ten  minutes. 

EFFECT  OF  THE  PROTEIN  HYDROLYSATES  ON  THE 
GROWTH  OF  STREPTOCOCCI. 

The  procedure  for  testing  the  effect  of  the  hydrolysates  on 
bacterial  growth  was  the  same  as  described  in  our  previous 
paper5.  One  c.c.  of  the  hydrolysate  was  added  to  the  sterile 
D.I.-G.S.  medium  and  the  whole,  after  suitable  inoculation,  incu- 
bated at  37° C.  for  24  hours.  Of  the  animal  proteins  tested,  only 
two,  casein  and  commercial  gelatin,  gave  a  definite  positive  stimu- 
lation to  the  growth  of  streptococci,  while  fibrin  and  lactalbumin 
gave  only  a  slight  activity.  Sulphuric  acid  hydrolysates  of  casein 
were  also  strongly  active.  Of  the  hydrolysates  of  the  proteins  of 
vegetable  origin,  we  found  only  two  to  support  the  growth  of 
streptococci, — namely,  edestin  and  yeast  protein. 

TABLE  I. 

Effect  of  Protein  Hydrolysates  on  Growth  of 
Streptococci  and  Yeast. 

Net  yeast  Bac- 

NO-  Hydrolysates  of  Animal  Proteins.    1  cc.  used.  growth  in  terial 

mm.  growth 

1.  Casein   (Purified)   HC1  hydrolysate 1.0  -f 

2.  "  "          H8SO«       "  0  + 

3.  "      (Technical)  HC1         «  1.0  + 

4.  Lactalbumin    0.6  ± 

5.  Fibrin    

6.  Serum  globulin   

7.  Serum  albumin 

8.  Beef -heart  (muscle)  protein   

9.  Egg  globulin    0                  

10.  Egg  albumin    


20  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

Net  yeast  Bac- 

No.  Hydrolysates  of  Animal  Proteins.    1   cc.  used.  growth  in  terjri 


11.  Vitellin  (egg  yolk)   .......  ..................... 

12.  Liver  globulin  (beef)   .......................... 

13.  Liver  albumin      "        .......................... 

14.  Gelatin  (commercial—  "Silver  Label"  brand)  .....        — 

15.  Gelatin  (prepared  from  bones  and  marrow)  ......        — 

16.  Gelatin  (prepared  from  bones  without  marrow)  .  .        — 

Hydrolysates  of  Vegetable  Proteins.  1  cc.  used. 

17.  Edestin  (hemp  seed)    ..........................        — 

18.  Gliadin    (wheat)    ..............................        — 

19.  Zein  (corn)   ...................................        — 

20.  Glutelin   (corn)    ............................... 

21.  Legumin  (peas)  ............................... 

22.  Vicilin  (peas)   ................................. 

23.  Legumelin  (peas)    ............................. 

24.  Oryzenin  (rice  ................................ 

25.  Hordein  (barley)  ..............................          0 

26.  Yeast  protein  ........................  .........       1.0  + 


-j-  denotes  growth. 

±  denotes  slight  growth. 

—  (under  Bacterial  column)  denotes  no  growth. 

0  (under  Yeast  column)  denotes  that  growth  was  same  as  control. 

—  (     "          "  "     )       "         inhibition. 

Quantitative  Results :  We  attempted  to  devise  a  method  for 
the  determination  of  the  amount  of  stimulation  of  the  growth 
of  streptococci  by  centrifuging  the  bacteria,  as  is  done  in  the 
method  for  yeast  described  by  Funk  and  Dubin.7  This  pro- 
cedure, however,  was  found  to  be  impractical,  due  to  the  dif- 
ficulty of  stopping  the  growth  of  bacteria,  and  also  because  of 
the  occasional  precipitation  of  substances  which  were  not  due 
to  bacterial  formation. 

A  better  method  for  comparative  results  was  devised,  based 
on  the  change  in  hydrogen  ion  concentration  due  to  the  fer- 
mentation of  the  glucose  in  the  D.I.-G.S.  medium  by  the  strepto- 
cocci. This  change  in  hydrogen  ion  concentration  was  deter- 
mined by  means  of  the  Sorenson8  indicator  method,  using  the 
Clark  and  Lubs9  indicator  set.  Cole  and  Jordan10  used  a  similar 
method  for  diagnosis,  by  fermentation  tests,  of  the  gonococcus, 
meningococcus  and  other  pathogenic  organisms. 

The  variation  in  hydrogen  ion  concentration,  in  all  of  our 
tests,  covered  a  range  from  a  PH  of  7.4  to  a  PH  of  4.0.  For  this 
test,  we  prepared  standard  solutions  consisting  of  mixtures  of 


LOUIS  FREEDMAN  21 

Na2HP04  and  NaH2P04  for  the  alkaline  range,  and  solutions 
consisting  of  mixtures  of  acetic  acid  and  sodium  acetate,  ac- 
cording to  Walpole11,  for  the  acid  range.  With  these  standard 
solutions,  we  used  the  following  indicators : 

Phenol  red,  for  PH  8.0 — 6.8 
Brom  thymol  blue,  for  PH  7.6—6.0 
Brom  cresol  purple,  for  PH  6.9 — 5.2 
Methyl  red,  for  PH  6.0—4.4 

Three-tenths  cc.  of  the  required  indicator  solution  was  added 
to  10  cc.  of  the  standard  solution.  The  colored  solutions  were 
then  put  into  uniform  test  tubes  and  the  tubes  sealed.  The 
tests  were  made  as  follows :  Duplicate  tubes  were  prepared,  each 
containing  9  cc.  of  the  D.I.-G.S.  medium  and  1  cc.  of  the  protein 
hydrolysate.  One  tube  was  inoculated  with  the  streptococci  and 
the  other  kept  sterile.  Both  tubes  were  incubated  together.  After 
twenty-four  hours,  0.3  cc.  of  the  indicator  necessary,  which  was 
determined  roughly  by  the  amount  of  bacterial  growth  formed, 
was  added  to  each  tube  and  the  resulting  color  compared  with  the 
standard  tubes. 

As  we  used  a  control  check  for  each  test,  we  found  this 
method  to  be  very  convenient  and  fairly  accurate  for  compara- 
tive results.  We  have  for  convenience  tabulated  the  results  ob- 
tained with  our  protein  hydrolysates  in  their  stimulating  action 
on  the  growth  of  streptococci,  in  tables  Ila  and  b. 

TABLE  II  (a) 

Quantitative  Effect  of  Protein  Hydrolysates  on  the 
Growth  of  Streptococci. 

PH  of  standard  D.I.-G.S.  solution  =  7.3 

PH 

No.  'Hydrolysates  of  Animal   Proteins.  Growth  after 

(1  cc.  used  in  each  test)  incubation 

1.  Casein  (purified)  HC1  hydrolysate +  5.8 

1  (a).  (sterile   control) —  7.3 

2.  Casein  (purified)  H2SO<  hydrolysate +  5.3 

2  (a).  (sterile  control) —  7.0 

3.  Casein  (technical)  HC1  hydrolysate +  5.8 

3  (a).  (sterile  control) —  7.3 

4.  Lactalbumin    ±  7.0 

4  (a).  (sterile  control)    —  7.3 

5.  Filbrin    —  7.3 

5  (a).        "         (sterile  control)    —  7.3 


22  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

TABLE  II.   (a)    (continued) 

PH 

No.                               Hydrolysates  of  Animal  Proteins.  Growth  after 

(1  cc.  used  in  each  test)  incubation 

6.  Serum  globulin —  7.3 

6  (a).        "                      (sterile  control)    —  7.3 

7.  Serum  albumin —  7.3 

7  (a).        "                      (sterile  control)    —  7.3 

8.  Beef-heart  (muscle)   protein —  7.2 

8  (a).                                               "      (sterile  control)..  —  7.3 

9.  Egg  globulin ±  7.0 

9  (a).      "                     (sterile  control)    —  7.3 

10.  Egg  albumin —  7.3 

10  (a).      "                     (sterile  control)    —  7.3 

11.  Vitellin  (egg  yolk)   —  7.3 

11  (a).                      "        "     (sterile  control) —  7.3 

12.  Liver  globulin   —  7.2 

12  (a).       "                      (sterile  control)  —  7.3 

13.  Liver  albumin    —  7.3 

13  (a).       "                      (sterile  control)   —  7.3 

14.  Gelatin  (commercial)   +  6.0 

14  (a).                                          (sterile  control) —  7.2 

15.  Gelatin  (prepared  from  bone  and  marrow) ....  —  7.2 

15  (a).          "  "  "         "      "          "       (ster- 

ile control)    —  7.3 

16.  Gelatin    (prepared    from    bone    without    mar- 

row)      —  7.2 

16  (a).    Gelatin  (prepared  from  bone  without  marrow) 

(sterile  control)   —  7.3 

TABLE  II  (b) 

PH  after 

No.                                 Hydrolysates  of  Vegetable  Proteins.  Growth  incubation 

17.  Edestin    +  6.5 

17  (a).                  (sterile  control)   —  7.3 

18.  Gliadin   —  7.0 

18  (a).                  (sterile  control)    —  7.1 

19.  Zein    —  7.3 

19  (a).      "      (sterile   control)    —  7.3 

20.  Glutelin   (corn)    —  7.0 

20  (a).                                 (sterile  control) —  7.2 

21.  Legumin    —  7.0 

21  (a).                    (sterile  control)  —  7.2 

22.  Vicilin   _  7.0 

22  (a).        "       (sterile  control)  —  7.2 


LOUIS  FREEDMAN  23 

TABLE  II.    (b)   (continued) 

PH  after 
No.  Hydrolysates  of  Vegetable  Proteins.  Growth  incubation 

23.  Legumelin    —  7.3 

23  (a).  (sterile   control)    —  7.3 

24.  Oryzenin  —  7.0 

24  (a).  (sterile  control)    —  7.2 

25.  Hordein    ±  7.0 

25  (a).  (sterile  control)    —  7.3 

26.  Yeast   protein    -f  4.9 

26  (a).        "  (sterile  control) —  7.3 

These  quantitative  results  confirm  our  first  results,  namely, 
that  only  two  of  our  animal  and  two  of  our  vegetable  proteins 
contain  the  growth  stimulating  vitamine  for  streptococci  in 
appreciable  amounts.  Of  the  proteins  whose  hydrolysates  con- 
tain the  active  substances,  yeast  protein  is  by  far  the  richest, 
with  casein  second,  commercial  gelatin  closely  following,  and 
edestin  containing  the  smallest  amount  of  the  active  substance. 

EFFECT  OF  PROTEIN  HYDROLYSATES  ON  GROWTH  OF  YEAST. 

The  protein  hydrolysates  as  described  above  were  tested  on 
yeast;  and,  with  the  exception  of  casein  and  yeast  protein,  all 
the  hydrolysates  either  failed  to  stimulate,  or  actually  inhibi- 
ted, the  growth  of  yeast  cells.  Even  the  action  of  casein  and 
yeast  protein  hydrolysates  were,  however,  practically  negative, 
the  net  increase  in  growth  of  yeast  cells  due  to  the  vitamine 
activity  of  the  hydrolysates  amounting  to  one  mm.  for  each 
of  these  proteins. 

These  results  are  not  at  all  surprising,  as  Vansteenberge12 
found  that  leucine,  tyrosine  and  asparagine  inhibited  the 
growth  of  yeast  cells  but  not  of  the  lactic  bacteria.  All  of  our 
hydrolysed  proteins  contain  large  amounts  of  leucine,  while  most 
of  them  contain  varying  amounts  of  tyrosine  and  aspartic  acid. 

A  close  analysis  of  the  results  obtained  with  these  proteins 
together  with  their  method  of  purification  and  their  physical 
structure  will,  if  anything,  tend  to  confirm  our  view  that  the 
growth-stimulating  action  is  due,  not  to  any  constituent  part 
of  the  protein  molecule,  but  to  the  tendency  of  the  protein  to 
adsorb  vitamine  and  hold  on  to  it  in  spite  of  all  attempts  at 
purification.  Thus,  yeast  protein,  the  hydrolysate  of  which  gave 
considerable  stimulation  to  the  growth  of  streptococci,  was  ob- 


24  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

tained  from  a  medium  which  we  know  to  be  very  rich  in  vitamine, 
particularly  the  newly  isolated  vitamine  D.  This  protein  was 
prepared  by  heating  the  filtered  autolyzed  yeast  until  the  protein 
had  coagulated.  As  this  protein  separates  in  a  form  similar  to 
milk  curd,  we  see  the  possibility  of  great  adsorption  of  vitamines. 
Although  this  protein  underwent  considerable  washing,  and  was 
suspended  in  a  dialyzing  bag  for  several  days,  it  still  retained 
most  of  the  vitamine  which  it  had  originally  adsorbed. 

The  same  is  true  of  casein.  This  protein,  which  is  precipi- 
tated by  means  of  dilute  acid  and  is  purified  by  repeatedly 
redissolving  and  reprecipitating,  separates  at  the  start  in  the 
form  of  a  coagulum,  and,  as  other  investigators  have  shown, 
has  great  adsorbing  powers.*  Consequently,  no  amount  of  re- 
dissolving  and  reprecipitating  will  entirely  free  it  from  vita- 
mines,  as  its  physical  structure  and  adsorbing  properties  remain 
the  same. 

With  gelatin,  we  have  a  somewhat  analogous  situation. 
Gelatin,  which  we  prepared  in  the  laboratory,  from  bones  with 
and  without  the  marrow,  was  entirely  freed  from  vitamines  by 
the  process  of  purification  described  by  Van  Name.14  This  con- 
sists, essentially,  in  precipitating  the  gelatin  in  absolute  alcohol, 
extracting  with  ether,  redissolving  the  protein  in  water,  concen- 
trating the  solution  to  a  jelly,  reprecipitating  in  absolute  alcohol 
and  again  extracting  with  ether.  Commercial  gelatin,  ("Silver 
Label"  brand)  however,  contains  enough  of  the  active  substance 
to  stimulate  the  .growth  of  streptoccoci,  as  we  have  shown  in 
tables  I  and  II.  It  seems  apparent  therefore  that  the  present 
commercial  process  for  purifying  gelatin  is  not  adequate  for 
freeing  this  protein  entirely  from  the  vitamines  contained  in 
the  bones  and  parts  of  the  hide  that  are  used.  In  this  connection 
it  might  be  mentioned  that  Boyer15  used  an  hydrochloric  acid 
extract  of  finely  divided  bones  for  the  cultivation  of  streptococci. 

With  edestin,  we  have  a  protein  which  was  prepared  in  a 
somewhat  different  manner.  We  prepared  this  protein  by 
extracting  the  ground  hempseed  with  five  percent  sodium 

The  following  far-reaching  statement  was  made  by  Prof.  F.  G.  Hopkins**  in  his 
Chandler  Lecture  given  at  Columbia  University,  1921.  "It  is  remarkable  what  a  considerable 
portion  of  the  vitamines  present  in  milk  is  adsorbed  by  precipitated  casein.  A  failure  to 
recognize  this  has  often  obscured  the  results  of  feeding  trials." 


LOUIS  FREEDMAN  25 

chloride  solution  at  60 °C.  The  solution  was  allowed  to  cool 
gradually  when  the  edestin  separated  as  a  crystalline  preci- 
pitate. It  was  purified  by  redissolving  in  warm  saline  solution 
and  again  cooling  the  solution.  Apparently,  edestin  also  has 
great  adsorbing  properties.  Osborne,  Wakeman  and  Ferry1  r> 
were  unable  to  free  edestin  entirely  from  vitamine. 

Regarding  the  proteins,  the  hydrolysates  of  which  showed 
no  growth  stimulating  activity,  a  review  of  their  methods  of 
preparation  and  purification  may  throw  some  light  on  this  point. 
Thus  the  prolamines,  e.g.,  wheat  gliadin,  zein  and  hordein  from 
cereals,  were  all  extracted  by  means  of  hot  alcohol,  the  alcoholic 
solutions  evaporated  to  small  volumes  and  the  protein  precipi- 
tated either  in  water  or  very  dilute  salt  solutions.  As  the  water 
soluble  vitamines  are  also  soluble  in  alcohol,  we  can  readily  see 
how  all  or  nearly  all  of  the  vitamines  would  remain  in  solution. 
Those  proteins,  such  as  the  albumins  and  globulins,  which  were 
salted  out  either  by  half  saturation  or  complete  saturation  with 
ammonium  sulphate  were  all  purified  by  dialysis,  and  we  know 
that  vitamines  are  dialysable.  Thus  it  is  possible  that  these 
albumins  and  globulins  have  very  little  adsorbing  properties,  and 
therefore  can  be  freed  from  vitamines  by  thorough  purification. 
The  same  apparently  holds  true  for  the  vegetable  globulins  with 
the  exception  of  edestin. 

The  glutelins  probably  were  free  from  vitamines  even  be- 
fore extraction  with  dilute  alkali,  as  they  were  obtained  from 
the  cereal  residues  after  the  extraction  of  the  gliadins  by  means 
of  alcohol.  At  any  rate,  the  vitamines,  if  present  would  prob- 
ably be  destroyed  by  the  continued  action  of  the  alkali. 

The  active  proteins,  from  published  analyses,  show  nothing 
in  common  that  cannot  be  shown  to  apply  to  the  other,  inactive, 
proteins,  so  that  we  can  safely  rule  out  the  question  of  amino 
acids  or  nitrogen  content.* 


*  McLeod  and  Wyon1T,  in  a  recently  published  report  on  the  "Supposed  importance  of 
vitamines  in  promoting  bacterial  growth,"  used  among  other  extracts,  alcoholic  extracts  of  the 
kidneys  of  the  guinea  pig,  and  they  believed  that  the  growth  was  due  to  vitamine  B,  although 
they  stated  that  the  amino  acid  content  of  the  extracts  was  also  a  factor.  They  also 
obtained  better  results  with  hydrolysed  casein  than  with  casein.  They  also  found  that 
"marmite,"  which  is  supposedly  rich  in  vitamines,  was  inactive  for  the  pneumococcus  and 
meningococcus,  and  that  charcoal,  devoid  of  vitamines,  when  suspended  in  their  bouillon 
cultures,  stimulated  the  growth  of  these  two  organisms.  The  part  of  the  work  of  these  authors 
dealing  with  charcoal  and  "marmite"  will  no  doubt  need  confirmation. 


26  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

In  summarizing  the  above  results,  three  points  stand  out  in 
explanation  of  the  growth  stimulating  power  of  the  hydrolysates 
of  proteins. 

1)  The  source  of  the  protein;  whether  it  is  obtained  from 
a  medium  rich  in  water-soluble  vitamine  of  the  B  type  such  as 
Vitamine  D. 

2)  The  physical  structure  of  the  protein  and  its  ability  to 
adsorb  vitamine. 

3)  The  method  of  purification. 

EXPERIMENTS  WITH  NEUTRAL  SODIUM  CASEINATE. 

If  these  active  substances  present  in  casein  and  three  of  the 
other  proteins,  as  described,  are  nothing  more  than  adsorbed 
vitamines,  and  if  the  protein  could  be  brought  into  solution  with- 
out subjecting  it  to  hydrolysis,  then  the  vitamine  would  also  go 
into  solution  and  could  be  removed  by  adsorbing  it  with  fullers 
earth  or  some  other  adsorbing  agent. 

To  test  this  theory,  we  prepared  a  solution  of  neutral 
sodium  caseinate  by  suspending  our  purified  casein  in  water,  and 
adding  five  percent  sodium  hydroxide  solution  until  the  casein 
had  practically  all  dissolved.  A  small  amount  of  insoluble  de- 
natured casein,  formed  by  the  action  of  the  alkali,  was  filtered 
off,  and  the  solution  was  then  made  neutral  to  litmus.  This 
neutral  sodium  caseinate  solution  (containing  about  5%  casein) 
was  shaken  for  three  hours  with  ten  grams  (50  grams  per  liter) 
of  fullers  earth. 

After  shaking,  the  fullers  earth  was  separated  from  the  solu- 
tion by  centrifugation,  as  filtration  was  very  slow,  due  to  the 
formation  of  an  emulsion,  and  the  supernatant  liquid  was  then 
filtered.  The  fullers  earth  was  repeatedly  washed  with  water 
and  centrifuged  to  remove  small  amounts  of  sodium  caseinate 
adhering  to  it,  pressed  out  on  a  suction  filter,  washed  with 
alcohol  and  dried.  This  fullers  earth  was  then  extracted  with 
baryta  and  worked  up  as  described  in  paper  I.5  The  resulting 
solution  was  made  up  to  a  volume  corresponding  to  a  ten  percent 
solution  of  the  original  casein,  adjusted  to  a  PH  of  7.4  and  ster- 
ilized. This  solution,  when  tested,  was  found  to  have  a  strong 
stimulating  effect  on  the  growth  of  streptococci,  but  had  no 
action  on  the  growth  of  yeast  cells. 

The  neutral  sodium  caseinate  solution,  after  shaking  with 


LOUIS  FREEDMAN  27 

fullers  earth,  was  subjected  to  hydrolysis  by  adding  concen- 
trated sulphuric  acid  until  a  30%  solution  was  obtained  and 
boiling  for  36  hours.  This  hydrolysate  failed  to  give  any  growth 
stimulating  activity  on  either  streptococci  or  yeast. 

It  is  therefore  apparent  that  most  or  all  of  the  vitamine  was 
removed  from  the  casein  as  described.  This  shows  that  the 
active  substance  in  the  protein  is  not  necessarily  a  constituent 
part  of  the  protein  molecule,  and  does  not  have  to  be  isolated  by 
breaking  up  the  protein.* 

SUMMARY  OF  CONCLUSIONS. 

1.  We  can  obtain  from  purified  casein,  commercial  gelatin, 
yeast  protein  and  edestin,  by  hydrolysis  of  these  proteins,  cer- 
tain substances  which  show  marked  growth  stimulating  activity 
on  hemolytic  streptococci.   Hydrolysates  of  purified  egg  globulin, 
lactalbumin  and  hordein,  show  traces,  whereas  hydrolysates  of 
the  other  proteins  examined  are  devoid  of  these  activating  sub- 
stances. 

2.  We  have  found  that  these  active  substances  are  not  a 
constituent  part  of  the  protein  molecule,  and  that  the  amount 
of  the  substances  present  in  the  protein  depends  on  the  physical 
and  adsorptive  properties  of  the  protein  and  the  method  and  de- 
gree of  purification. 

3.  The  properties  of  these  active  substances,  as  obtained 
from  the  proteins  described  above,  show  that  they  are  probably 
related  to  if  not  identical  with  the  water-soluble  vitamines  ob- 
tained from  brewers'  yeast,  particularly  vitamine  D. 

4.  We  have  described  a  method  for  determining  the  amount 
of  these  active  substances  by  the  use  of  Sorensen's  indicator 
method,  which  is  based  on  the  change  in  hydrogen  ion  concentra- 
tion due  to  the  fermentative  action  of  the  bacteria.    This  method 
is  advocated  for  comparative  results  only,  and  not  as  a  direct 
quantitative  method. 


*  The  experiments  with  neutral  sodium  caseinate,  just  described,  may  be  of  practical 
importance  in  that  they  may  lead  to  a  method  of  preparing  a  vitamine-free  casein.  The 
casein  thus  obtained  gives  positive  tests  for  all  the  color  reactions  known  for  the  various 
»mino  acids,  while  its  nitrogen  content  is  the  same  as  that  of  the  original  casein  from  which 
it  was  prepared.  Further  work  is  now  in  progress  on  this  problem. 


\ 


28  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 

BIBLIOGRAPHY  TO  PART  II. 

1.  Bainbridge,  F.  A.,  J.  Hyff.,  1911,  11,  341. 

2.  Robinson,  H.  C.,  and  Rettger,  L.  F.,  J.  Bad.,  1918,  3,  209. 

3.  Mueller,  J.  Howard,  Proc.  Soc.  Exp.  Biol.  Med.,  1921,  18,  225. 

4.  Goy,  Pierre  C.,  rend.  Acad.  Sci.,  1921,  172,  242. 

5.  Freedman,  L.,  and  Funk,  C.,  J.  Metabolic  Research,  1922,  1,  457. 

6.  Funk,  C.,  and  Dubin,  H.  E.,  J.  Biol.  Chem.,  1921,  48,  437. 

7.  Funk,  C.,  and  Dubin,  H.  E.,  J.  Biol.  Chem.,  1920,  44,  487. 

8.  Sorensen,  Bioc/iem.  Ztsc/i.,  1909,  21,  253. 

9.  Clark  and  Lubs,  J.  Bad.,  1917,  2,  109  and  191. 

10.  Cole  and  Jordan,  J.  Path,  and  Bact.,  June,  1917. 

11.  Walpole,  G.  S.,  Biochem.  J.,  1914,  8,  636. 

12.  Vansteenberge,  P.,  Ann.  Inst.  Past.,  1917,  31,  601. 

13.  Hopkins,  F.  G.,  J.  Ind.  Eng.  Chem.,  1922,  14,  67. 

14.  Van  Name,  W.  G.,  J.  Exp.  Med.,  1897,  2,  117. 

15.  Boyer,  Louis,  C.  r.  soc.  de  biol.,  1918,  81,  229. 

16.  Osborne,  T.  B.,  Wakeman,  A.  J.,  and  Ferry,  E.  A.,  J,  Biol.  Chtm.,  1919, 

39,  35. 

17.  McLeod,  J.  W.,  and  Wyon,  G.  A.,  J.  Path,  and  Bact.,  1921,  24,  205. 


LOUIS  FREEDMAN  29 


ACKNOWLEDGMENT. 

I  wish  to  express  my  sincere  appreciation  to  Professors 
W.  J.  Gies  and  E.  G.  Miller,  Jr.,  of  the  Department  of  Biological 
Chemistry,  for  their  valuable  advice  and  criticism  in  the  prep- 
aration of  this  dissertation.  I  also  wish  to  express  my  gratitude 
to  Dr.  Casimir  Funk,  of  the  Department  of  Biological  Chemistry, 
who  suggested  the  problem  and  under  whose  painstaking  guid- 
ance this  work  was  done. 

I  also  take  this  opportunity  to  express  my  acknowledgment 
and  thanks  to  the  Hon.  Herman  A.  Metz,  who  extended  many 
facilities  to  me  in  this  work  and  gave  me  the  opportunity  of  com- 
pleting my  dissertation  while  engaged  in  research  in  his 
laboratory. 


30  NUTRITION  FACTORS  IN  YEAST  AND  BACTERIA 


VITA. 

Louis  Freedman  was  born  in  New  Haven,  Conn.,  January 
28,  1894.  He  graduated  from  the  New  Haven  High  School  in 
1912  and  entered  the  Sheffield  Scientific  School  of  \ale  University 
the  same  year.  During  freshman  year,  he  was  the  recipient  of  a 
Connecticut  High  School  scholarship.  He  received  the  degree  of 
Ph.  B.  in  1915,  after  which  he  spent  two  years  in  the  Yale  Gradu- 
ate School,  from  which  he  received  the  degree  of  Master  of 
Science.  During  his  graduate  course,  he  was  the  holder  of  both 
a  graduate  and  an  industrial  fellowship.  From  1917  to  1919  he 
was  employed  by  the  Calco  Chemical  Company  as  research  chem- 
ist in  Pharmaceuticals.  During  the  war,  he  was  engaged  in 
special  research  work  on  high  explosives  for  the  United  States 
Government.  He  matriculated  in  Columbia  University  in  Sep- 
tember, 1920,  and  is  at  present  associated  with  the  Research 
Laboratory  of  H.  A.  Metz,  New  York  City. 


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